Pulse-shaping for Multiphoton FRET Microscopy In Vivo Multiphoton microscopy has proven to be an invaluable tool for imaging in scattering tissue and live animals. However, quantitative multiphoton studies of protein-protein interactions have been hindered by the expense and limited spectral range of the typical laser sources available for multiphoton microscopy. Broadband titanium-sapphire laser sources offer a stunning range of excitation wavelengths in a single laser source, allowing simultaneous excitation of multiple fluorophores. However, with broadband excitation comes a lack of excitation selectivity. Pulse-shaping offers an ideal solution for providing efficient and selective multiphoton excitation, with the added benefit of reduced photobleaching. This proposal aims to combine pulse-shaping with quantitative multiphoton FRET microscopy in vivo, introducing an exciting new tool to the research community. This new technology will permit rapid and quantitative imaging of many commonly-used two photon fluorophores and FRET pairs, including many fluorescent proteins. We will initially demonstrate multiplex fluorescence excitation and detection of commonly-used fluorescent proteins and FRET pairs in solution, employing spectral-unmixing and FRET stoichiometry algorithms to obtain quantitative measures of relative free and interacting protein concentrations. We will then employ the optimized pulse-shapes in live-cell imaging applications to provide quantitative measures of cell membrane receptor signaling. We will compare the pulse-shaping-based technology with one-photon FRET stoichiometry and quantitative multiphoton FRET imaging employing fluorescence lifetime methodology (FRET-FLIM). Compared to FRET-FLIM and current multiplex multiphoton imaging based on laser-tuning we anticipate orders of magnitude enhancement in image acquisition speed, enabling multiphoton FRET microscopy in vivo. In optimizing the pulse-shaping methodology for multiphoton FRET we will also gain insight into photobleaching and photodamage reduction mechanisms that will be widely applicable to other multiphoton imaging techniques. PUBLIC HEALTH RELEVANCE: Pulse-shaping for Multiphoton FRET Microscopy In Vivo The proposed work will develop versatile technology to permit rapid and quantitative imaging and analysis of multiple protein interactions via multiphoton fluorescence resonance energy transfer (FRET), revolutionizing our ability to investigate cell signaling in vivo. The fundamental optical methods and algorithms developed will be transferred readily to other investigators, enabling widespread use of this technology in biomedical studies of normal development and human disease. In optimizing the pulse-shaping methodology for multiphoton FRET applications we will also gain insight into methods to reduce photobleaching and photodamage that will be widely applicable to other multiphoton imaging techniques.